Spatial localization of CD16a at the human NK cell ADCC lytic synapse

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by FcγRIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.


Isolation of primary NK cells
For assays that required primary NK cells, we utilized viably frozen PBMCs (a gift from Andrew Ward's lab).NK cells were isolated using an EasySep TM Human NK Cell Isolation Kit (StemCell Tech) according to the instructions provided in the kit.Freshly isolated NK cells were utilized immediately in ADCC assays (see below).

CD16-SNAP plasmid
The CD16-SNAP construct (Fig. S1) was synthesized and cloned into a transposon plasmid without reporter by Vector Builder.

Cell line generation
The NK-92 CD16-SNAP cell line was constructed using the PiggyBac (PB) system.Approximately 3x 10 6 NK-92 (ATCC) cells were co-nucleofected with a CD16-SNAP plasmid and the hyPBase plasmid carrying the transposase (VectorBuilder) at ratio of 3:1, respectively, using a Lonza P3 Primary Cell kit in approximately 100 µL medium and a Lonza 4D-nucleofector device with the program CA137.Post-nucleofection, cells were resuspended in complete medium and allowed to recover for 48 hours at 37˚C in 5% CO2.Then half of the medium was removed and replaced with fresh complete medium.After 96 hours post-transfection, half of the medium was removed, and cells were resuspended again in a total of 10 mL of complete medium.After approximately 1-week post-transfection, cells were stained with an a-CD16-AF488 (3G8 Biolegend, 1:20) antibody for 1 hour on ice and sorted using a Sony MA900 or Thermofisher Scientific Bigfoot cell sorter.Gating was performed on cells with fluorescence intensity greater than parental NK-92.After sorting, cells were placed in a 96 well plate U-bottomed plate in 200 µL of complete medium with IL-2 and expanded.

Flow cytometry
Confirmation of CD16a expression was performed by flow cytometry.Briefly, ~100,000 cells were placed in an Eppendorf tube and pelleted at 200 x g at 4°C for 5 minutes.Cells were then resuspended in 500 µL of PBS containing 1% (w/v) BSA.Cells were then washed twice with fresh, cold PBS with 1% BSA and then finally stained with an a-CD16-AF488 antibody (3G8 Biolegend, 1:20) for 1 hour at 4°C.Cells were then washed twice with PBS with 1% BSA and resuspended in 200 µL for analysis on a CytoFLEX S (Beckman Coulter).Data analysis was performed using FlowJo.

ADCC assay
NK-92 CD16-SNAP ADCC activity was measured using an Agilent xCELLigence Real-Time Cell Analysis (RTCA) SP impedance instrument that was setup in a stationary 37˚C incubator with 5% CO2.On the first day of the experiment, SKOV-3 cells were detached using TrypLE TM Express medium, quenched with complete medium, and counted. 1 x 10 6 cells were removed and pelleted at 250 x g for 10 minutes before being resuspended in complete Roswell Park Memorial Institute 1640 Medium (RPMI) at 1 x 10 5 cells/mL.Meanwhile, 50 µL of complete RPMI was added to an E-Plate 96 PET plate (Agilent) and background impedance was measured.Then, 5,000 cells (50 µL) were added to each well and allowed to sit at room temperature for 30 minutes.The plate was then transferred to the instrument and impedance measured every 15 minutes for ~24 hours to allow cells to attach to the plate and establish baseline cellular impedance.The next day, 50 µL of Trastuzumab (Selleckchem) was prepared in complete RPMI medium and was added to yield a final concentration of 1 -0.0016 µg/mL.While the antibody was allowed to bind, NK cells, NK-92, and/or NK-92 CD16-SNAP cells were counted and pelleted at 150 x g for 10 minutes.Cells were then resuspended at 5 x 10 5 cells/mL and 50 µL added to yield a final effector to target ratio of 5:1.The plate was then allowed to sit at room temperature for 30 minutes before being transferred into the instrument, and impedance was measured every 15 minutes for 24 hours.ADCC activity was determined using the Agilent xCELLigence software and curves were plotted using Prism (GraphPad).

Sample preparation for confocal laser scanning microscopy
Cover glasses (Corning, 18mm #1.5) were coated with 0.1% (w/v) PLL, a-CD18 antibody (5 µg/mL in PBS), or a-CD16/a-CD18 antibodies (5 µg/mL in PBS) for 30 minutes at room temperature.Cover glasses were then washed 3 times with PBS before plating NK-92 CD16-SNAP cells (200 µL at ~1 x 10 6 cells/mL) and placed in a humidified incubator at 37°C and 5% CO2 for 30 minutes.Medium was then carefully removed by pipetting, and the sample was washed with PBS.Cells were then fixed with 4% PFA (w/v) for 15 minutes, permeabilized with 0.4% (w/v) Triton X-100 for 3 minutes and fixed again with 4% PFA for 15 minutes.Fixed cells were washed with PBS, quenched with NH4Cl (50 mM) for 5 minutes, and washed 3 times with PBS.Samples were then blocked with Image-IT (Invitrogen) for 30 minutes and washed 3 times with PBS.
Labeling of SNAP-tagged CD16a was performed with 1 µM AF647-SNAP (New England Biolabs) in PBS (0.5% w/v BSA, 1 mM DTT) for 50 minutes.Labeling solution was then removed, and samples were washed 2 times with PBS and allowed to wash in fresh PBS for 30 minutes.
PBS was then replaced with PBS containing 5% (w/v) BSA for ~2 hours.Samples were then incubated with an a-pCD3 antibody (BD Bioscience, clone K25-407.69,AF488, 1:50) at 4°C overnight.Subsequently, samples were washed 3 times with PBS, post-fixed with 4% PFA for 3 minutes, fixative quenched with NH4Cl (50 mM) for 5 minutes, and finally washed 3 times with PBS before mounting the sample with Prolong Diamond (Invitrogen) for imaging.
Cover glasses were first cleaned by sonication for 5 minutes in acetone, followed by 5 minutes of sonication in absolute ethanol, and a final sonication in MiliQ water.After each sonication step, the cover glasses were dried with a stream of nitrogen gas.After cleaning by sonication, the cover glasses were further cleaned by oxygen plasma in a Solarius plasma cleaner with a power of 20 W and a process pressure of 1.6 torr for 15 minutes before being attached to a 6 channel stickyslide (Ibidi VI 0.4).After assembly of the channel slide, liposomes (50µL at 4 mg/mL) were deposited into each channel and incubated at room temperature for 20 minutes.The channels were then washed 10 times with PBS.SLBs were incubated with 100 µM NiCl2 containing 1%(w/v) BSA for 20 minutes at room temperature.The samples were then washed 5 times with PBS and incubated with His-tagged HER2 (10 µg/mL; Acro Biosystems) and His-tagged ICAM-1 (1 µg/mL; Acro Biosystems) for 60 minutes at 37°C, after which the samples were washed 4 times with PBS.

Immunological synapse formation on SLBs
SLBs used for the preparation of NK cell immune synapses were prepared as described above, and subsequently incubated with either Trastuzumab (Selleckchem) at 10 µg/mL in PBS for 30 minutes at room temperature or PBS alone as a control, followed by three washes with PBS.
Meanwhile, NK cells were counted, and 1 x 10 6 cells were removed and pelleted at 100 x g for 10 minutes.Cells were then resuspended in serum-free RPMI and 100 µl were added to each channel of the prepared slide and incubated at 37°C in 5% CO2 for 30 minutes.Medium was then removed, and samples were washed with PBS, fixed with 4%(w/v) PFA for 15 minutes, permeabilized with 0.4%(v/v) Triton X-100 for 3 minutes, and then fixed again with 4%(w/v) PFA for 15 minutes.Fixed cells were then washed with PBS, fixative was quenched with NH4Cl (50 mM) for 5 minutes, and the cells again washed 3 times with PBS.Samples were then blocked with Image-IT (Invitrogen) for 30 minutes and washed 3 times with PBS.Labeling of SNAP-tagged CD16a was performed with 2 µM AF647-SNAP (New England Biolab) in PBS containing 0.5%(w/v) BSA and 2 mM DTT for 50 minutes.Labeling solution was then removed, and samples were washed 2 times with PBS then allowed to wash in fresh PBS for 30 minutes.PBS was then replaced with PBS containing 5%(w/v) BSA for ~2 hours.Samples were next incubated with an a-pCD3 antibody (BD Bioscience, clone K25-407.69,AF488) at 4°C overnight.Subsequently, samples were washed 3 times with PBS, post-fixed with 4%(w/v) PFA for 3 minutes, fixative was quenched with 50 mM NH4Cl for 5 minutes, the samples finally washed 3 times with PBS and fresh PBS was added before imaging.

Preparing samples for fluorescence recovery after photobleaching (FRAP) sample preparation, collection and analysis
SLBs were subjected to FRAP to determine bilayer fluidity.Samples were first prepared as described above.Subsequently, an a-Her2 antibody (R&D systems research grade trastuzumab biosimilar, AF488, 10µg/mL, 1:100) was added and incubated for 30 minutes at room temperature.
Samples were then washed 3 times with PBS before proceeding to data collection.Fluorescently labeled SLBs were imaged on a Zeiss LSM 880 laser scanning confocal microscope using perfect focus with the following conditions: excitation at 488 nm (7% power) with a 493-630 nm emission detection window, 63x / 1.20 NA C-Apochromat water objective, 0.77 µs pixel dwell time, 800V gain offset, and 0.099 µm pixel size.Images were acquired every 30 seconds for 30 scans.After two scans, a region of interest (ROI) of ~25 µm 2 was photobleached (100% power, 10 µs pixel dwell time, for a single scan) and subsequent fluorescence recovery was recorded.

Confocal image collection
Confocal images of NK cells stained for SNAP-CD16 and pCD3 were acquired on a Zeiss LSM 780 laser scanning confocal microscope under the following conditions: excitation at 488nm and 633nm (10% power) emission with detection windows of 493-628 nm and 638-755 nm, respectively, a 63X / 1.40NA plan-apochromat oil objective with a pixel dwell time of 4.08 µs, line averaging of 2, pixel size of 0.09 µm, and a master gain of 800V.Z-stacks of ~2 µm were collected with a step size of 200 nm for each cell to cover the height of the immunological synapse.The in focus z-plane was then used for quantification.

MINFLUX sample preparation
Samples for MINFLUX data collection were stained for SNAP-CD16 as described above for confocal sample preparation.After removal of excess SNAP-AF647 staining solution, cells were labeled with phalloidin (Invitrogen, AF488, 1:200) for 60 minutes and washed 3 times with PBS.
Before mounting, 150 nm diameter gold fiducials (AUROlite TM Au/TiO2, STREM) were added to the samples for 5 minutes and then washed to remove unbound beads away 3 times with PBS.

Supplemental Figure 3 :
Data analysis workflow for MINFLUX nanoscopy collections.Flow chart describing the data analysis pipeline used to analyze MINFLUX data.Raw MINFLUX data was first filtered, and the center of clusters identified provide the likely fluorophore localization.

Fluorophore
localizations were used to determine nearest neighbor distance.A second set of analysis of fluorophore localization was then used to determine if the data is clustered using Ripley's H.If the data was clustered, then clusters were identified using DBSCAN.The identified clusters were then counted.Secondary analysis was performed on localizations not in clusters, and pairs of localizations identified.Then the nearest neighbor distance between isolated pairs performed.SupplementalFigure 4: Mean localization precision of MINFLUX data.(A) Representative mean localization precision of a data set acquired with cells on glass coated with a-CD16 and a-CD18 antibodies (4,146 trace centers).(B) Representative mean localization precision of a data set acquired with cells on glass coated with a-CD18 antibody (4,744 trace centers).(C) Representative mean localization precision of a data set acquired with cells on glass coated with PLL (5,119 trace centers).(D) Representative mean localization precision of a data set acquired with cells on an SLB without antibody (2,564 trace centers).(E) Representative mean localization precision of a data set acquired with cells on an SLB opsonized with Trastuzumab (3,510 trace centers).Supplemental Figure 5: Supported lipid bilayers present mobile antigens.(A) Representative image series acquired during FRAP experiment.(B) Raw fluorescence intensity from FRAP data.(C) Normalized fluorescence intensity presented in B. Supplemental Figure 6: NK-92 CD16-SNAP activate on SLB with Trastuzumab for increased number of clusters.Comparison of the number of clusters identified by DBSCAN between NK cell synapses on SLBs without Trastuzumab (-Traz) and with Trastuzumab (+ Traz).MINFLUX microscopy data were collected across at least three independent experiments with a total of 12 cells analyzed.** p = 0.0047.